We have continued to purify and characterize a novel class of pluripotential hematopoietic stem cells (PHSC) that lack c-Kit (c-Kit-neg), the receptor for stem cell factor (SCF). This is in contrast to previously characterized PHSC populations which express c-Kit (c-Kit-pos). We have demonstrated that the c-Kit-neg PHSC have delayed reconstituting activity and can give rise to c-Kit-pos PHSC when transplanted into irradiated mouse recipients. Taken together, we have hypothesized that steady state hematopoiesis is supported by PHSC that express c-Kit, and that c-Kit-neg PHSC represent a quiescent population of PHSC that are recruited into the actively contributing pool of c-Kit-pos stem cells. To better understand the mechanisms which regulate this maturation pathway, we have further purified this stem cell population and evaluated its growth and differentiation in vivo and in vitro. We have developed a surrogote in vivo transplantation assay in RAG-2 dificient mice that allows us to measure stem cell activity after one month rather than 8-10 months. We have determined that c-Kit-neg PHSC can be induced to proliferate and differentiate on M2B109 and OP9 stromal cell lines in vitro. We are evaluating whether we can induce the expression of c-Kit on c-Kit-neg PHSC by co-culture on stromal cell lines and whether the c-kit-pos cells have PHSC activity which will allow us to define the mechanism(s) which regulate the c-Kit maturation pathway. In other studies, we have continued our efforts to develop more efficient vectors and methods to transfer genes into primitive hematopoietic stem cells. In this regard, there are no current methods to efficiently deliver therapeutic genes to hematepoietic cells in vivo. Therefore, we have developed a procedure to inject gene therapy vectors directly into the intrafemoral space of mice pretreated with 5-flourouracil. We found that we could deliver gene therapy vectors to stem/progenitor cells by direct intrafemoral injection of adenoviral and retroviral vectors in vivo. Using this methodology we have corrected a monogenic disorder (SCID) by direct intrafemoral injection of a retroviral vector that expresses JAK-3 kinase. The molecular events that regulate lineage commitment and terminal differentiation of pluripotential hematopoietic stem cells (PHSC) are largely unknown. In this regard, we have evaluated the role of CCAAT enhancer-binding (C/EBP) proteins in hematopoietic cell growth and differentiation using the C/EBPa knock-out mouse model. We have found that fetal liver (FL) cells from C/EBPa knock out mice show an enhanced proliferative potential and concomitant decreased differential potential in response to hematopoietic growth factors. We have derived growth factor-dependent myelomonocytic cell lines directly from C/EBPa -/- mice. Preliminary evidence demonostrates that mice transplanted with C/EBPa -/- FL cells develop leukemias after long latency. Taken together, C/EBPa may act as a tumor suppressor in hematopoietic progenitors and contribute to the development of myeoid leukemias. We have discovered that Evi-9 (zinc finger transcription factor) is required for B cell differentiation (in collaboration with Neal Copeland's Laboratory). B cell maturation is arrested prior to the expression of B220 in in FL cells from Evi-9 null animals (Evi-9 null animals are moribund 24 hours after birth due to unknown causes). B cell maturation is similarly arrested in the bone marrow of mice transplanted with Evi-9 null fetal liver cells.